Tuberculosis and disseminated Mycobacterium avium cause significant morbidity and death in individuals with Human Immunodeficiency Virus (HIV) infection. Globally, tuberculosis is a leading cause of death for patients infected with HIV. The emergence of M. tuberculosis strains resistant to two or more anti-tuberculosis drugs have compromised control strategies. M. avium Complex (MAC) causes disseminated infection and increased mortality in up to 40 percent of patients with AIDS. There is an urgent need to develop new, potent anti-mycobacterial drugs. Mycolic acids are integral and unique parts of the mycobacterial cell wall and the biosynthetic enzymes represent attractive targets for the development of novel drugs. Towards that goal, we are using a multi-disciplinary approach employing mycobacterial genetics, biochemistry, x-ray crystallography, molecular biology, organic chemistry and animal models to develop novel inhibitors. We have discovered a gene, named inhA, which was found to be a primary target to isoniazid (INH) and ethionamide (ETH). Point mutations within the structural gene, or overexpression of inhA confers INH- and ETH- resistance in mycobacteria. Biochemical studies reveled that inhA encodes an NADH-specific enoyl-acyl carrier protein (ACP) reductase which prefers long chain fatty acids as substrates, consistent with its role in mycolic acid biosynthesis. The three dimensional structure of resistant and sensitive forms of InhA were determined. We demonstrated that INH is a pro-drug which, upon activation by a catalase-peroxidase forms a covalent adduct to NADH bound on the InhA enzyme. Armed with the knowledge of InhA structure and function, we have generated a series of long-chain fatty acid analog inhibitors that contain a triple bond between C2 and C3: the 2-alkynoic acids (KOAs). The KOAs inhibit the InhA enzyme and have activity against MAC, and against both INH- susceptible and INH-resistant M. tuberculosis. Furthermore, we have screened a combinatorial library of compounds against the InhA enzyme and identified two new classes of compounds with activity against InhA that inhibit M. tuberculosis and MAC growth. In this proposal, we intend to expand upon these successes and continue to elucidate the key targets of mycolic acid biosynthesis with the aim of developing novel drugs against M. tuberculosis and M. avium.